1. Field of the Invention
The present invention relates to dihydrodipicolinate synthase and dihydrodipicolinate reductase derived thermophilic Bacillus bacteria and genes coding for them.
2. Related Art
In the production of L-lysine by fermentation, strains isolated from nature or artificial mutants thereof have been used in order to improve the productivity. Many artificial mutant strains that produce L-lysine are known, and many of them are aminoethylcysteine (AEC) resistant strains and belong to the genus Brevibacterium, Corynebacterium, Bacillus or Escherichia. Further, various techniques have been disclosed for increasing the amino acid production, for example, use of a transformant obtained by using recombinant DNA (U.S. Pat. No. 4,278,765).
Dihydrodipicolinate synthase (abbreviated as xe2x80x9cDDPSxe2x80x9d hereinafter) is an enzyme that synthesizes dihydrodipicolinate through dehydration condensation of aspartic acid semialdehyde and pyruvic acid, and this reaction serves as an entrance of the branching into the L-lysine biosynthesis system in the biosynthesis of amino acids of aspartic acid type. Further, dihydrodipicolinate reductase (abbreviated as xe2x80x9cDDPRxe2x80x9d hereinafter) is known as one of important enzymes of the L-lysine biosynthesis system, which catalyzes the reaction in which the dihydrodipicoliniate generated in the aforementioned reaction is reduced to generate piperidinedicarboxylic acid.
As for microorganisms belonging to the genus Escherichia or Corynebacterium, the gene (dapA) which codes for DDPS has been cloned, and the nucleotide sequence thereof has also be determined. As for the genus Escherichia, methods for producing L-lysine by enhancing DDPS have been disclosed in Japanese Patent Laid-open Publication (Kokai) No. 56-18596/1981, U.S. Pat. No. 4,346,170 and Applied Microbiology and Biotechnology, 15, pp.227-331 (1982). Furthermore, a method for producing L-lysine using an Escherichia bacterium introduced with DDPS derived form Corynebacterium bacteria, which is known not to suffer feedback inhibition by L-lysine, has been disclosed in Korean Patent Publication No. 92-8382.
The gene coding for DDPR (dapB) has also been already obtained from the genus Escherichia (Bouvier, J. et al., J. Biol. Chem., 259, 14829 (1984)) and the genus Corynebacterium (Journal of Bacteriology, 175 (9), 2743-2749 (1993)). Furthermore, there has also been disclosed a method for improving production rate and productivity of L-lysine by enhancing the dapB gene derived from Corynebacterium bacterium together with the aspartokinase gene (WO96/40934).
The current mainstream of the L-lysine production is the fermentative production by using a coryneform bacterium or an Escherichia bacterium. In this production, however, enzymes required for the fermentation may be inactivated or the production bacteria may be killed due to temperature increase in the medium during the fermentation, and thus it is necessary to cool the medium during the fermentation.
By the way, enzymes and proteins produced by thermophilic bacteria are generally stable at elevated temperatures, and also stable against pH variation or organic solvents. Therefore, applications thereof as diagnostic regents, industrial catalysts and so forth have been highly developed. If it becomes possible to produce L-lysine by fermentation at elevated temperatures by utilizing such stability and durability of enzymes derived from thermophilic bacteria, the cooling of the medium becomes unnecessary, and therefore the cost for cooling during the fermentation can be reduced. Moreover, if fermentation at elevated temperatures is realized, it is expected that the reaction rate may also be improved
The present invention is accomplished in view of the aforementioned technical aspect, and its object is to obtain genes of the L-lysine biosynthesis system of thermophilic bacteria and thereby provide novel methods for producing L-lysine.
The inventors of the present invention assiduously studied in order to achieve the aforementioned object. As a result, they successfully isolated genes that coded for DDPS and DDPR from Bacillus methanolicus, which is one of the thermophilic Bacillus bacteria, and determined the nucleotide sequences of these genes. Thus, they accomplished the present invention.
That is, the present invention provides the followings.
(1) A protein defined in the following (A) or (B):
(A) a protein which has the amino acid sequence of SEQ ID NO: 2 shown in Sequence Listing, or (B) a protein which has an amino acid sequence of SEQ ID NO: 2 shown in Sequence Listing including substitution, deletion, insertion, addition or inversion of one or several amino acids, and has dihydrodipicolinate synthase activity.
(2) A DNA which codes for a protein defined in the following (A) or (B):
(A) a protein which has the amino acid sequence of SEQ ID NO: 2 shown in Sequence Listing, or
(B) a protein which has an amino acid sequence of SEQ ID NO: 2 shown in Sequence Listing including substitution, deletion, insertion, addition or inversion of one or several amino acids, and has dihydrodipicolinate synthase activity.
(3) The DNA according to (2), which is a DNA defined in the following (a) or (b):
(a) a DNA which has a nucleotide sequence comprising at least the nucleotide sequence of the nucleotide numbers 1 to 924 in SEQ ID NO: 1 shown in Sequence Listing; or
(b) a DNA which is hybridizable with a nucleotide sequence comprising at least the nucleotide sequence of the nucleotide numbers 1 to 924 in SEQ ID NO: 1 shown in Sequence Listing under a stringent condition, and codes for a protein having dihydrodipicolinate synthase activity.
(4) The DNA according to (3), wherein the stringent condition is a condition in which washing is performed at 60xc2x0 C., 1xc3x97SSC and 0.1% SDS.
(5) A protein defined in the following (C) or (D):
(C) a protein which has the amino acid sequence of SEQ ID NO: 4 shown in Sequence Listing, or
(D) a protein which has an amino acid sequence of SEQ ID NO: 4 shown in Sequence Listing including substitution, deletion, insertion, addition or inversion of one or several amino acids, and has dihydrodipicolinate reductase activity.
(6) A DNA which codes for a protein defined in the following (C) or (D):
(C) a protein which has the amino acid sequence of SEQ ID NO: 4 shown in Sequence Listing, or
(D) a protein which has an amino acid sequence of SEQ ID NO: 4 shown in Sequence Listing including substitution, deletion, insertion, addition or inversion of one or several amino acids, and has dihydrodipicolinate reductase activity.
(7) The DNA according to (6), which is a DNA defined in the following (c) or (d):
(c) a DNA which has a nucleotide sequence comprising at least the nucleotide sequence of the nucleotide numbers 1 to 798 in SEQ ID NO: 3 shown in Sequence Listing; or
(d) a DNA which is hybridizable with a nucleotide sequence comprising at least the nucleotide sequence of the nucleotide numbers 1 to 798 in SEQ ID NO: 3 shown in Sequence Listing under a stringent condition, and codes for a protein having dihydrodipicolinate reductase activity.
(8) The DNA according to (7), wherein the stringent condition is a condition in which washing is performed at 60xc2x0 C., 1xc3x97SSC and 0.1% SDS.
(9) A microorganism which is introduced with the DNA according to (2) or the DNA according to (6) or the both in a form that allows expression of a protein encoded by each DNA.
(10) A method for producing L-lysine, which comprises culturing the microorganism according to (9) in a medium to produce and accumulate L-lysine in the medium, and collecting the L-lysine from the medium.
In the present invention, the expression of xe2x80x9cto have dihydrodipicolinate synthase activityxe2x80x9d is used to mean to have an activity for catalyzing the reaction of dehydration condensation of aspartic acid semialdehyde and pyruvic acid to generate dihydrodipicolinate. The expression of xe2x80x9cto have dihydrodipicolinate reductase activityxe2x80x9d is used to mean to have an activity for catalyzing the reaction of reduction of dihydrodipicolinate to generate piperidinedicarboxylic acid, and to mean that the activity is higher at 50xc2x0 C. than at 37xc2x0 C.
According to the present invention, there are provided DDPS and DDPR, which are involved in the L-lysine biosynthesis, with excellent heat resistance, and genes coding for them.
Hereafter, the present invention will be explained in detail.
The DNA of the present invention can be obtained through selection of clones containing a DDPS gene or a DDPR gene based on recovery of auxotrophy in mutant strains of microorganisms deficient in DDPS or DDPR used as an index from a gene library of thermophilic Bacillus bacteria, for example, Bacillus methanolicus. 
The method for obtaining the DNA of the present invention will be explained below.
 less than 1 greater than  Production of Gene Library of Bacillus methanolicus 
A gene library of Bacillus methanolicus can be produced, for example, as follows. First, the total chromosome DNA is prepared by the method of Saito et al. (Saito, H. and Miura, K., Biochem. Biophys. Acta, 72, 619-629, (1963)) or the like from a wild-type strain of Bacillus methanolicus, for example, Bacillus methanolicus PB1 (NCIMB13113) strain, and partially digested with a suitable restriction enzyme, for example, Sau3AI and so forth to obtain a mixture of various fragments. If the degree of the digestion is controlled by adjusting digestion reaction time and so forth, restriction enzymes of a wide range can be used.
Subsequently, the digested chromosome DNA fragments are ligated to vector DNA autonomously replicable within Escherichia coli cells to produce recombinant DNA. More specifically, a restriction enzyme producing the same end nucleotide sequence as the restriction enzyme used for the digestion of the chromosome DNA is allowed to act on the vector DNA to fully digest the vector and cleave it. Then, the mixture of the chromosome DNA fragments and the cleaved vector DNA obtained as described above are mixed, and a DNA ligase, preferably T4 DNA ligase, is allowed to act on the mixture to obtain recombinant DNA.
By using the obtained recombinant DNA, Escherichia coli, for example, the Escherichia coli JM109 strain and so forth, is transformed, and a gene library solution can be prepared from culture of obtained transformants. The transformation can be performed by, for example, the method of D. M. Morrison (Methods in Enzymology, 68, 326 (1979)) and the method in which recipient cells are treated with calcium chloride so as to increase the permeability of the cells for DNA (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)). Electroporation was employed in the examples mentioned below.
Examples of the vector include, for example, pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF100, pMW119, pMW118, pMW219, pMW218, pSTV28 and so forth. In addition, phage vectors can also be used. For example, since a chloramphenicol resistance gene is contained in pSTV28, only transformants harboring the vector or the recombinant DNA can be grown by using a medium containing chloramphenicol when that vector is used.
Examples of the method for collecting the recombinant DNA from the cells after the transformants are cultured include the alkali SDS method and so forth.
 less than 2 greater than  Screening of Clones Containing DDPS Gene or DDPR Gene
By using a gene library solution of Bacillus methanolicus obtained as described above, a mutant strain of microorganism deficient in DDPS or DDPR is transformed, and clones showing recovery of auxotrophy are selected. Examples of such a mutant strain of microorganism deficient in DDPS include the Escherichia coli AT998 (CGSC4548) strain, which is deficient in DDPS. Since the Escherichia coli AT998 strain is deficient in the DDPS gene, it cannot grow in a minimal medium that does not contain diaminopimelate. On the other hand, a transformant strain thereof which harbors the DDPS gene derived from Bacillus methanolicus can grow in the minimal medium, because the gene functions. Therefore, a DNA fragment containing the DDPS gene can be obtained by selecting a transformant strain that can grow in the minimal medium and collecting recombinant DNA from the strain.
Examples of the mutant strain of microorganism deficient in DDPR include the Escherichia coli AT999 strain (CGSC 4549), which is deficient in the DDPR gene. Since the Escherichia coil AT999 strain is deficient in the DDPR gene, it shows only a slow growth rate even in a complete medium such as L medium if it does not contain diaminopimelate. On the other hand, a transformant strain thereof which harbors the DDPR gene derived from Bacillus methanolicus shows normal growth, because the gene functions. Further, the Escherichia coli AT999 cannot grow in a minimal medium, whereas a transformant strain thereof which harbors the DDPR gene derived from Bacillus methanolicus can grow in the minimal medium, because the gene functions. Therefore, a DNA fragment containing the DDPR gene can be obtained by selecting a transformant strain which can grow in the minimal medium and collecting recombinant DNA from the strain.
By extracting an inserted DNA fragment from the obtained recombinant DNA and determining nucleotide sequence of the fragment, the nucleotide sequence and the amino acid sequence of the DDPS gene or the DDPR gene and DDPS or DDPR can be determined.
Determination of nucleotide sequences, digestion and ligation of DNA and so forth may be attained by those methods commonly used for gene cloning (detailed in, for example, Sambrook J., Fritsch, E. F. and Maniatis, T., 1989, Molecular Cloning: A Laboratory Manual, Second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. etc.). They can also be conducted according to instructions attached to reagents such as restriction enzymes and kits.
The DDPS gene of the present invention codes for DDPS that has the amino acid sequence of SEQ ID NO: 2 shown in Sequence Listing. Specific examples of the DDPS gene of the present invention include DNA that has the nucleotide sequence of SEQ ID NO: 1. Further, the DDPS gene of the present invention may have a nucleotide sequence including replacement of codons for each amino acid with equivalent codons, so long as the sequence codes for the same amino acid sequence as the amino acid sequence shown as SEQ ID NO: 2.
Further, the DDPS gene of the present invention may be one coding for a protein which has an amino acid sequence of SEQ ID NO: 2 including substitution, deletion, insertion, addition or inversion of one or several amino acids, or one coding for a protein which has DDPS activity. The term xe2x80x9cseveralxe2x80x9d amino acids used herein means preferably 1-50 amino acids, more preferably 1-10 amino acids. Homology between the DDPS gene of the present invention and a known DDPS gene of Bacillus subtilis (B. subtilis) is 65.9% on the basis of the nucleotide sequence, and 64.8% on the basis of the encoded amino acid sequence.
The DDPR gene of the present invention codes for DDPR that has the amino acid sequence of SEQ ID NO: 4 shown in Sequence Listing. Specific examples of the DDPR gene of the present invention include DNA that has the nucleotide sequence of SEQ ID NO: 3. Further, the DDPR gene of the present invention may have a nucleotide sequence including replacement of codons for each amino acid with equivalent codons, so long as the sequence codes for the same amino acid sequence as the amino acid sequence shown as SEQ ID NO: 4.
Further, the DDPR gene of the present invention may be one coding for a protein which has an amino acid sequence of SEQ ID NO: 4 including substitution, deletion, insertion, addition or inversion of one or several amino acids, or one coding for a protein which has DDPR activity. The term xe2x80x9cseveralxe2x80x9d amino acids used herein means preferably 1-40 amino acids, more preferably 1-10 amino acids. Homology between the DDPR gene of the present invention and a known DDPR gene of Bacillus subtilis (B. subtilis) is 66.7% on the basis of the nucleotide sequence, and 67.5% on the basis of the encoded amino acid sequence.
DNA that codes for the substantially same protein as DDPS or DDPR as described above is obtained by modifying the nucleotide sequence, for example, by means of the site-directed mutagenesis method so that one or more amino acid residues at a specific site should contain substitution, deletion, insertion, addition or inversion. DNA modified as described above may also be obtained by conventionally known mutation treatments. Such mutation treatments includes a method for treating DNA coding for DDPS or DDPR in vitro, for example, with hydroxylamine or the like, and a method for treating a microorganism, for example, a bacterium belonging to the genus Escherichia, harboring DNA coding for DDPS or DDPR with ultraviolet irradiation or a mutagenizing agent usually used for the mutation treatment such as N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine (NTG) and nitrous acid.
The substitution, deletion, insertion, addition, or inversion of nucleotides as described above also includes mutation (mutant or variant) which naturally occurs, for example, due to the individual difference or the difference in species or genus of the microorganism that harbors DDPS or DDPR.
Such DNA coding for substantially the same protein as DDPS or DDPR is obtained by expressing DNA having mutation described above in an appropriate cell, and investigating the DDPS or DDPR activity of the expression product. DNA coding for substantially the same protein as DDPS or DDPR is also obtained by isolating DNA which is hybridizable with a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 1 or 3 shown in Sequence Listing or a part thereof, for example, a probe which can be prepared from the nucleotide sequence of SEQ ID NO: 1 or 3 by PCR, under a stringent condition, and codes for a protein having DDPS or DDPR activity.
The xe2x80x9cstringent conditionxe2x80x9d referred to herein is a condition under which a so-called specific hybrid is formed, and a non-specific hybrid is not formed. It is difficult to clearly define this condition by using numerical values. However, for example, the stringent condition includes a condition under which DNA""s having high homology, for example, DNA""s having homology of not less than 40% are hybridized with each other, and DNA""s having homology lower than the above are not hybridized with each other. Alternatively, the stringent condition is exemplified by a condition under which DNA""s are hybridized with each other at a salt concentration corresponding to an ordinary condition of washing in Southern hybridization, i.e., 60xc2x0 C., 1xc3x97SSC, 0.1% SDS, preferably 0.1xc3x97SSC, 0.1% SDS (see, for example, Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second edition, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. etc.).
Such genes, which are hybridizable under the condition as described above, includes those having a stop codon generated in a coding region of the genes, and those having no activity due to mutation of active center. However, such mutants can be easily removed by ligating the genes with a commercially available activity expression vector, and measuring the DDPS or DDPR activity. The DDPS activity can be measured by, for example, the method of Yugari et al. (Yugari Y. and Gilvarg C., Journal of Biological Chemistry, 240, 4710 (1962)). Specifically, for example, the DDPS activity can be measured by allowing a reaction of 100 xcexcl of 500 mM imidazole hydrochloride (pH 7.5), 100 xcexcl of 20 mM aspartic acid semialdehyde (which can be synthesized by the method described in Black S. and Write N., Journal of Biological Chemistry, 213, 51 (1955)), 100 xcexcl of 20 mM sodium pyruvate and 100 xcexcl of an enzyme solution in a total volume of 1 ml and measuring increase of absorbance at 270 nm.
The DDPR activity can be measured by, for example, the method of Tamir et al. (Tamir H. and Gilvarg C., Journal of Biological Chemistry, 249, 3034 (1974)). Specifically, for example, the DDPR activity can be measured by allowing a reaction of 100 xcexcl of 500 mM imidazole hydrochloride (pH 7.5), 100 xcexcl of dihydrodipicolinic acid, 100 xcexcl of NADPH and 100 xcexcl of an enzyme solution in a total volume of 1 ml and measuring decrease of absorbance at 340 nm.
Because the nucleotide sequences of the genes which code for DDPS and DDPR derived from Bacillus methanolicus were elucidated by the present invention, the DNA sequence which codes for DDPS or DDPR can be obtained from Bacillus methanolicus gene library by hybridization using an oligonucleotide probe prepared based on each of the sequences. DNA sequences which code for the enzymes can also be obtained by amplification from Bacillus methanolicus chromosome DNA by PCR (polymerase chain reaction) using oligonucleotide primers prepared based on the aforementioned nucleotide sequences.
 less than 3 greater than  Application of DDPS Gene and DDPR Gene
The DDPS gene and the DDPR gene of the present invention can be used for production of DDPS and DDPR. That is, DDPS can be produced by introducing DNA containing the DDPS gene into a suitable host cell, and culturing the obtained transformant to allow expression of the DNA. DDPR can be produced by introducing DNA containing the DDPR gene into a suitable host cell, and culturing the obtained transformant to allow expression of the DNA. The produced DDPS protein or DDPR protein can be collected from the culture and purified by techniques used for usual purification of proteins such as salting out, solvent precipitation, gel filtration chromatography and ion exchange chromatography.
The DDPS gene and the DDPR gene can also be utilized for breeding of L-lysine producing bacteria. By introducing the DDPS gene, the DDPR gene or the both into a microorganism, L-lysine biosynthesis is enhanced and thus L-lysine producing ability is improved.
Examples of the host cell into which the DDPS gene or the DDPR gene is introduced include Escherichia bacteria such as Escherichia coli, coryneform bacteria such as Brevibacterium lactofermentum, Bacillus bacteria such as Bacillus methanolicus and so forth. Examples of the vector used for introducing the DDPS gene or the DDPR gene into these hosts include, as for Escherichia bacteria, those mentioned above. As for coryneform bacteria, the following vectors can be mentioned. There are indicated microorganisms which harbors each vector, and accession numbers thereof at international depositories are shown in the parentheses, respectively.
pAJ655
Escherichia coli AJ11882 (FERM BP-136)
Corynebacterium glutamicum SR8201
(ATCC39135)
pAJ1844
Escherichia coli AJ11883(FERM BP-137)
Corynebacterium glutamicum SR8202
(ATCC39136)
pAJ611 Escherichia coli AJ11884 (FERM BP-138)
pAJ3148 Corynebacterium glutamicum SR8203
(ATCC39137)
pAJ440 Bacillus subtilis AJ11901 (FERM BP-140)
These vectors are obtained from deposited microorganisms as follows. Cells collected at the logarithmic growth phase are lysed with lysozyme and SDS to give a lysate, from which a supernatant solution is obtained by centrifugation at 30,000xc3x97g. Polyethylene glycol is added to the supernatant solution to perform fractional purification by means of cesium chloride-ethidium bromide equilibrium density gradient centrifugation.
In order to introduce a plasmid into E. coli to transform it, there may be used a method in which recipient cells are treated with calcium chloride so as to increase the permeability of the cells for DNA (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)) and so forth.
Examples of the vector for Bacillus bacteria include, for example, pUB110, pHY300PLK, pHV1248, pE194, pC194, pBC16, pSA0501, pSA2100, pAM77, pT181, pBD6, pBD8, pBD64, pHV14 and so forth.
Transformation of coryneform bacteria may be performed by the electric pulse method (Sugimoto et al., Japanese Pat. Publication Laid-Open No. 2-207791/1990). Transformation of Bacillus bacteria may be performed by a method of making host sells into the protoplast or spheroplast followed by introducing recombinant DNA into the DNA-recipient cells (Chang, S. and Choen, S. N., Molec. Gen. Genet., 168, 111 (1979); Bibb, M. J., Ward, J. M. and Hopwood, O. A., Nature, 274, 398 (1978); Hinnen, A., Hicks, J. B. and Fink, G. R., Proc. Natl. Acad. Sci., USA, 75, 1929 (1978)).
The DDPS gene or the DDPR gene to be introduced may be introduced into a host with a promoter proper to the gene, or the structural gene ligated to another promoter may be introduced. Examples of such a promoter include lac promoter, trp promoter, trc promoter, tac promoter, PR promoter and PL promoter of xcex phage, tet promoter, amyE promoter, spac promoter and so forth.
L-lysine can be produced by culturing a microorganism introduced with the DDPS gene or the DDPR gene or the both genes and having L-lysine producing ability in a medium to produce and accumulate L-lysine in the medium, and collecting the L-lysine from the medium.
Although medium and culture conditions can suitably be selected according to kind of the host microorganism to be used, usual media can be used, which contain a nitrogen source, inorganic ions and other organic trace amount nutrients as required.
As the carbon source, there can be used saccharides such as glucose, lactose, galactose, fructose and hydrolysate of starch, alcohols such as glycerol and sorbitol, organic acids such as fumaric acid, citric acid and succinic acid and so forth.
When a methanol assimilating bacterium such as Bacillus methanolicus is used as the microorganism of the present invention, methanol can be preferably used as the carbon source.
As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, aqueous ammonia and so forth can be used.
As the inorganic ions or sources thereof, a small amount of potassium phosphate, magnesium sulfate, iron ions, manganese ions and so forth may be added. As a trace amount organic nutrient, it is desirable to add a suitable amount of required substances such as L-homoserine and vitamin B1, yeast extract and so forth as required.
The culture is performed under conditions suitable for growth of a microorganism to be used. In general, it is preferably performed for 16 to 72 hours under an aerobic condition, and the culture temperature is controlled to be 20 to 45xc2x0 C., and pH to be 5-8.5 during the culture. For adjusting pH, inorganic or organic acidic or alkaline substances, ammonia gas and so forth can be used. Further, when a thermophilic bacterium is used as a host, it can be cultured at a culture temperature of 42 to 60xc2x0 C.
Collection of L-lysine from the culture can usually be carried out by using a combination of known techniques such as techniques using ion exchange resins, precipitation methods and so forth.